Somatic adrenal stem cells are believed to reside in the periphery of the adrenal cortex throughout life for organ maintenance. Herein, we used the side population (SP) phenomenon to enrich for these progenitors, which made up to 0.01–0.64% of the total cell count. Microarray analysis revealed an expression profile of SP cells, which clearly differed from that of non-SP cells. However, a promising adrenal specific stem cell marker could not be identified.In vitro, SP cells could be maintained in long-term culture, whereas non-SP cells did not proliferate. After 4 weeks of culturing, immunohistochemistry revealed the expression of steroidogenic enzymes such as 3β-HSD, StAR, and P450SCC, suggesting spontaneous differentiation. Interestingly, the quantity of SP cells was significantly diminished inPbx1haploinsufficient mice, suggesting a stem cell deficit. By contrast, the subcapsular zone of ACTH-deficientTpit^−/−mice was significantly wider compared with wild-type adrenals (Tpit^−/−259±10.7 vsTpit^+/−100±12.3%;P<0.01). Accordingly, the number of SP cells in these mice was significantly higher (Tpit^−/−0.45±0.16 vsTpit^+/−0.13±0.04%;P<0.004). ACTH treatment of these animals reverted the subcapsular zone width and the SP fraction back to normal (130±10.2%;P=0.33 and 0.09%), providing indirect evidence for a stem cell ‘arrest’ inTpit^−/−mice and the role of ACTH in adrenocortical stem cell modulation and differentiation.